Method for production of limulus-positive glycolipid, the limulus-positive glycolipid, and composition containing the limulus-positive glycolipid

ABSTRACT

It has been found that a  limulus -positive glycolipid is present in xanthan gum derived from  Xanthomonas , which has been commercially available and eaten for many years, and this was purified, and it has been found that this  limulus -positive glycolipid has an immunopotentiation effect. A method for safely and inexpensively producing the  limulus -positive glycolipid containing an immunopotentiator at high concentrations is provided. The method for producing the  limulus -positive glycolipid of the present invention comprises extracting the  limulus -positive glycolipid from xanthan gum. A  limulus -positive glycolipid composition containing the  limulus -positive glycolipid can be used for various applications such as pharmaceuticals, pharmaceuticals for animals, quasi drugs, cosmetics, foods, functional foods, feedstuff and bath agents.

This application is a divisional application of U.S. application Ser.No. 12/308,997, filed Dec. 31, 2008, and claims the right of priorityunder 35 U.S.C. §119 based on Japanese Patent Application No.2006-194965 filed Jul. 14, 2006, which is hereby incorporated byreference herein in its entirety as if fully set forth herein.

TECHNICAL FIELD

The present invention relates to a method for producing a xanthan gumlimulus-positive glycolipid which is an immunopotentiator and is safewhen added in pharmaceuticals, quasi drugs, cosmetics, functional foods,feedstuff, fertilizers and bath agents for plants and animals such asmammals including humans (specifically domestic animals, pet animals,etc.), birds (specifically farmed chicken, pet birds, etc.), amphibiananimals, reptiles, fish (specificallypet fish, etc.) and invertebrates,and a composition containing a limulus-positive glycolipid.

BACKGROUND ART

It is an urgent problem to establish disease prevention and therapeuticmethods including infection prevention technology for mammals includinghumans (specifically domestic animals, pet animals, etc.), birds(specifically farmed chicken, pet birds, etc.), amphibian animals,reptiles, fish (specifically pet fish, etc.) and invertebrates.Furthermore, in order to achieve this, the methods using no chemicals,without environmental pollution, without producing resistant bacteriaand without accumulation in the human body are strongly required. Thepresent inventors have already found for the above problems that theimmunopotentiators derived from natural products safely achieve diseaseprevention and therapeutic effects (Non-patent Document 1). As oneexample thereof, it is possible to use lipopolysaccharide obtained fromPantoea agglomerans which is a resident microbiota with wheat flour(Non-patent Document 1). It has been known that a limulus-positiveglycolipid has a potent immunoenhancing activity (Non-patent Document2). This limulus-positive glycolipid includes so-calledlipopolysaccharide. It has been known that lipopolysaccharide is a majorcomponent of an outer wall of a cell of gram-negative bacteria as wellas a major component of Coley's vaccine and has a potentimmunopotentiation activity (Non-patent Document 3).

The present inventors have found that a limulus-positive glycolipid ispresent in wheat flour, a part thereof is lipopolysaccharide of aresident microbiota with wheat and they strongly potentiate innateimmunity (Non-patent Document 4). And, the above two potentiate innateimmunity safely and potently and exhibit protective and therapeuticeffects on various diseases including infectious diseases byadministering them percutaneously or orally (Non-patent Document 5).Furthermore, the present inventors have reported that a fermented wheatextract which is a novel immunopotentiator, not only in which thecontent of lipopolysaccharide derived from Pantoea agglomerans isincreased, but also which contains the component derived from wheat byfermenting wheat flour with Pantoea agglomerans which is a residentmicrobiota with wheat flour, exerts an effect for infection preventionas a safe and trouble-free natural material in place of antibioticsubstances or chemicals in the fields of animal industry andaquaculture.

A basic structure of lipopolysaccharide is composed of lipid referred toas lipid A and various types of sugars (polysaccharide) covalently boundthereto. A portion subsequent to lipid A is composed of R core which hasa relatively uniform structure in related species and a subsequentO-antigen polysaccharide portion which has a different structuredepending on the species (Non-patent Document 7). The O-antigen has arepeating structure of the same oligosaccharide characteristic for LPS(lipopolysaccharide) (Non-patent Document 1). Therefore,lipopolysaccharide generally forms a mixture having multiple molecularweights. It has also been known that lipopolysaccharide has a differentstructure depending on the microorganism which it is derived from. Forexample, lipopolysaccharide derived from Salmonella andlipopolysaccharide derived from Escherichia coli are different instructure and also in biological activity. However, in general, it isnot easy to determine the structure of lipopolysaccharide. Thus, detailsof the structure and function of lipopolysaccharide in many gramnegative bacteria have not been known. Thus, it has been described thatlipopolysaccharide has a novel structure based on its functionaldifference.

Moreover, it has been demonstrated in recent studies thatlipopolysaccharide activates innate immunity via TLR4 (Non-patentDocument 6). It has been found that the lipid A moiety oflipopolysaccharide is essential for binding to TLR4 and a polysaccharidemoiety greatly affects efficiency of intracellular signal transductionof TLR4. From the above, it is speculated that the difference incellular response to lipopolysaccharide is attributed to a structuraldifference.

It is important in establishing the usefulness of lipopolysaccharide toconfirm that percutaneously or orally administered lipopolysaccharide issafe and trouble-free. Thus, the gram negative bacteria used forproducing and fermenting foods since ancient times have gained focus.That is, if limulus-positive glycolipid, inter alia lipopolysaccharideis present in the gram negative bacteria used for producing foods orprovided for human consumption with fermented products, this factconfirms eating experience for limulus-positive glycolipid orlipopolysaccharide. This is a finding which strongly shows thatpercutaneously or orally administered lipopolysaccharide is safe andtrouble-free, as well as encouraging the development of new health careproducts such as cosmetics and foods, and pharmaceuticals using thesesubstances.

[Non-patent Document 1] Chie Kohchi et al., “Innate Immunity RegulatoryAction of Fermented Wheat Extract,” New Food Industry (2006) Vol. 48, p.19-27.

[Non-patent Document 2] Ulmer, A. J. et al., “Lipopolysaccharide:Structure, Bioactivity, Receptors, and Signal Transduction,” Trends inGlycoscience and Glycotechnology, (2000) Vol. 14, p. 53-68.

[Non-patent Document 3] Starnes, C. O., “Coley's Toxins in Perspective,”Nature, (1992) Vol. 357, p. 11-12.

[Non-patent Document 4] Nishizawa, T. et al., Chem. Pharm. Bull., (1992)Vol. 40, p. 479-483.

[Non-patent Document 5] Hiroyuki Inagawa et al., “Therapeutic andPreventive Effect of Lipopolysaccharide (LPSw) having MacrophageActivation Action and Derived from Wheat of Various Diseases,”Biotherapy, (1991) Vol. 5, p. 617-621.

(Non-patent Document 6) Kiyoshi Takeda et al., “Toll-like Receptors inInnate Immunity.,” International Immunology, Vol. 17, p. 1-14.

[Non-patent Document 7] Seikagaku Jiten 2nd Edition (1990), Tokyo KagakuDojin, p. 1949.

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

In order to orally or percutaneously administer to humans or animals,safety supported with eating experience is required. For example, aceticacid bacterium used for making a vinegar is gram negative bacterium, itsmicrobial cell component is included in its fermented product, and thuscan be said to have been eaten for many years. Pantoea agglomeranssupplies folic acid required for lactic acid growth of rye breads, and aconsiderable amount of its microbial cell component is ingested when ryebread is eaten.

Xanthan gum is polysaccharide produced from Xanthomonas campestris whichis gram negative bacterium, and is composed of mannose, glucose andglucuronic acid. In xanthan gum, β-1,4-linked glucose is a main chain,and in a side chain, two molecules of mannose and glucuronic acid arebound for every other glucose residue in the main chain. Xanthan gum hashigh viscosity, is stable in relation to acids, salts and heat, and iswidely used as a thickener for foods. It was introduced in Japan about30 years ago, and currently 1,300 tons or more of xanthan gum isannually consumed. From these, xanthan gum is a product fermented withgram negative bacterium, having been eaten for many years.

In this application, it has been found that limulus-positive glycolipidwhich had not been known until now is present in xanthan gum derivedfrom Xanthomonas, which has been commercially available and eaten formany years, and this was purified, and it has been found that thislimulus-positive glycolipid has an immunopotentiation effect.

Means for Solving the Problems

The method for producing the limulus-positive glycolipid of the presentinvention comprises extracting the limulus-positive glycolipid fromxanthan gum.

The limulus-positive glycolipid of the present invention is obtained bythe above method for producing the limulus-positive glycolipid.

The limulus-positive glycolipid composition of the present inventioncontains the above limulus-positive glycolipid.

The limulus-positive glycolipid composition is a pharmaceutical, apharmaceutical for animals, a quasi drug, a cosmetic, a food, afunctional food, a feedstuff or a bath agent.

Effect of the Invention

According to the present invention, it is possible to obtaintrouble-free and safe limulus-positive glycolipid because it has beenfound that limulus-positive glycolipid is present in xanthan gum whichhas been commercially available and eaten for many years. Thislimulus-positive glycolipid can be used for various applications such ashealth care products and pharmaceuticals containing this because thelimulus-positive glycolipid has been purified and it has been found thatthis has an immunopotentiation effect.

The present specification incorporates contents disclosed in thespecification and drawings in Japanese Patent Application No.2006-194965 which is a basis of priority of this application.

BEST MODES FOR CARRYING OUT THE INVENTION

Extraction of limulus-positive glycolipid from xanthan gum

EXAMPLES

1) Extraction of Limulus-Positive Glycolipid from Xanthan Gum

Xanthan gum (1.00 g) (Dainippon Sumitomo Pharma Co., Ltd.) was added toone liter of phosphate buffered saline (PBS), and homogenized using apolytron at a scale of 6 for 10 minutes. A polymyxin B immobilized resin(5 ml) (Affiprep polymyxin support supplied from BIO-RAD, California)was added to the resulting PBS solution of 0.1% (w/v %) xanthan gum, andthe solution was stirred using a magnetic stirrer for 4 hours. It hasbeen known that polymyxin B is bound to a molecule such as lipid Ahaving a high lipid-solubility. After stirring, the solution wastransferred to a 50 ml centrifuge tube, which was then centrifuged at2,000 rpm at room temperature for 10 minutes. After completing thecentrifugation, the supernatant was discarded, and Affiprep polymyxinsupport was collected as a precipitate. 10 ml of PBS was added to theprecipitate, stirred, and a precipitate was collected by thecentrifugation at 2,000 rpm at room temperature. 5 ml of an aqueoussolution of 0.02N sodium hydroxide was added to the precipitate, stirredfor one minute, and centrifuged at 2,000 rpm at room temperature for 10minutes. After completing the centrifugation, the supernatant wascollected in another container. 5 ml of an aqueous solution of 0.02Nsodium hydroxide was added again to a precipitate, stirred for oneminute, and centrifuged at 2,000 rpm at room temperature for 10 minutes.After completing the centrifugation, two supernatants were combined.Immediately, 1 ml of 1 mol/liter (M) tris hydrochloride, pH 7.0 wasadded to neutralize the supernatants. It was identified by aphenol-sulfuric acid method that the resulting solution contained asugar. Thus, this solution was made a limulus-positiveglycolipid-containing extracted solution from xanthan gum.

The limulus-positive glycolipid was measured using Endospecy (suppliedfrom Seikagaku Corporation; limulus test which does not react withβ-1,3-glucan), and about 4 mg of the limulus-positive glycolipid waspresent. Because about 5 mg of the limulus-positive glycolipid wascontained in xanthan gum, about 80% was yielded.

2) Measurement of Immunopotentiation Effect of Limulus-PositiveGlycolipid

The limulus-positive glycolipid was added to RAW264.7 which was acultured murine macrophage lineage cell line, and the production ofnitrogen monoxide from the cells was measured.

Because RAW264.7 cells were weakly adherent cells, the cells werecollected by pipetting from a culture flask and the cell concentrationwas adjusted to 8×10⁵ cells/ml using a medium. A cell suspension (100μl) was transferred to each well of a 96-well flat bottomed plate, whichwas used for a test after 6 hours when the cells were almost adhered.

A concentration of the limulus-positive glycolipid was adjustedequivalent to 4000 ng/ml of a lipopolysaccharide-concentration inPantoea agglomerans. A serial dilution for 5 scales by 10 times wasfurther performed. Each diluted solution was further diluted twice withthe medium, and each diluted solution was further diluted twice with themedium containing 40 μg/ml polymyxin B. The resulting solution was usedas a preparation for adding to the well in which the cells had beenadded.

Simultaneously, lipopolysaccharide (LPSx) purified from Xanthomonascampestris was also examined. 100 μl of each preparation was added toeach well to which the cells had been previously added in the 96-wellflat bottomed plate. The cells were cultured in a 5% carbon dioxide gasincubator at 37° C. for 20 hours. After completing the culture, 50 μl ofthe culture supernatant was collected. The amount of nitrous acid whichwas a metabolite of nitrogen monoxide in the medium was measured usingGriess reagent according to standard methods.

Measurement results were shown in Tables 1 and 2. The limulus-positiveglycolipid extraction solution from xanthan gum at concentrations of 100ng/ml or higher exhibited production of NO from RAW264.7 cells. An NOproduction ability of LPSx was 22 times higher than that of thelimulus-positive glycolipid when compared at 7.0 μM (the amount of thelimulus-positive glycolipid required to be added for obtaining 7.0 μM ofa nitrous acid concentration was 2.8 ng/ml whereas the amount of LPSxwas 61.2 ng/ml, 61.2/2.8=22). In polymyxin B-adding groups, the NOproduction was not observed by both samples at 100 ng/ml, but wasobserved at 1,000 ng/ml. Thus, it was identified that they had astructure bound to polymyxin B.

TABLE 1 Ability of limulus-positive glycolipid from xanthan gum toinduce NO production from RAW264.7 cells Nitrous acid concentration(μM)Added concentration Limulus-positive glyco- (ng/ml) LPSx lipid fromxanthan gum 0 1.61 ± 0.22 1.54 ± 0.22 0.1 1.69 ± 0.24 1.39 ± 0.34 1 3.84± 0.52 1.98 ± 0.49 10 10.65 ± 0.65  2.28 ± 0.29 100 12.36 ± 0.50  8.28 ±0.11 1000 14.13 ± 0.42  13.69 ± 0.27  Each measurement value was shownby mean ± standard deviation of 4 examples.

TABLE 2 Ability of limulus-positive glycolipid from xanthan gumpretreated with polymyxin B to induce NO production from RAW264.7 cells.Nitrous acid concentration(μM) Added concentration Limulus-positiveglyco- (ng/ml) LPSx lipid from xanthan gum 0 1.91 ± 0.36 1.98 ± 0.34 0.12.28 ± 0.29 2.21 ± 0.21 1 2.06 ± 0.26 1.76 ± 0.27 10 1.84 ± 0.22 1.76 ±0.33 100 3.47 ± 0.25 2.43 ± 0.15 1000 5.84 ± 0.45 9.17 ± 0.50 Eachmeasurement value was shown by mean ± standard deviation of 4 examples.

From the above results, it has been found that the limulus-positiveglycolipid from xanthan gum has action to induce the NO production frommacrophages, but is different from lipopolysaccharide (LPSx) obtainedfrom Xanthomonas microbial cells in biological activity. These resultsshow that the structural difference is present between them.

3) Measurement of Molecular Weight of Limulus-Positive GlycolipidExtracted from Xanthan Gum

A molecular weight of the limulus-positive glycolipid extracted fromxanthan gum was examined. Thus, 4 μg of limulus-positive glycolipid wasmixed with a sample buffer, and sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) using tricine was performed. Afterelectrophoresis, silver staining by Sil-Best Stainkit (Cat. No.30642-41, Nacalai Tesque, Japan) was performed for the purpose ofvisualizing the molecule of lipopolysaccharide. Prestained ProteinMarker, Broad Range (Premixed Format) (Cat. No. 7708L, NEW ENGLANDBiolabs) was used as a molecular weight size marker. As a result, theLPSx sample showed many ladder-like bands in 2,000 to 4,500 Daltons (Da)and 30 to 80 kDa. In the limulus-positive glycolipid extracted fromxanthan gum, the bands were detected in 2,000 to 4,000 Da. The molecularweights of the limulus-positive glycolipid extracted from xanthan gumare 2,000 to 4,000 Da whereas the molecular weights of LPSx were 2,000to 4,500 Da and 30 to 80 kDa. This result shows that a structuraldifference is present between them.

4) Antigenic Difference Between Limulus-Positive Glycolipid Extractedfrom Xanthan Gum and LPSx

If there is a structural difference between the limulus-positiveglycolipid extracted from xanthan gum and lipopolysaccharide (LPSx)obtained from Xanthomonas microbial cells, the difference of antigensoccurs and the reactivity of antibodies is likely different. Thus, thelimulus-positive glycolipid extracted from xanthan gum was stained witha monoclonal antibody against LPSx. The limulus-positive glycolipid wasdropped on a polyvinylidene fluoride (PVDF) membrane (supplied fromBIO-RAD), and subsequently non-specific reactions were blocked byincubating the PVDF membrane in PBS containing 3% bovine serum albumin(BSA) at room temperature for 30 minutes (blocking). Subsequently, thePVDF membrane was washed five times with Tris buffered saline (TBS: 20mM Tris HCl, pH 7.5, 150 mM NaCl) containing 0.05% Tween 20. Afterwashing, 5 ml of the medium containing an antibody specific tolipopolysaccharide obtained from the Xanthomonas microbial cell as aprimary antibody was added and reacted at room temperature for 60minutes.

-   (the monoclonal antibody specific for Xanthomonas lipopolysaccharide    was made as follows. Heated and killed Xanthomonas bacteria were    mixed with complete Freund's adjuvant, and 1×10⁸ bacteria per mouse    were administered intraperitoneally to BALB/c mice. The    intraperitoneal administration was repeated three times at intervals    of two weeks. Three days after the final administration, cells were    collected from the spleen, and fused with myeloma cells (P3U1) using    50% polyethylene glycol (average molecular weight: 1000). The fused    cells were suspended in HAT medium containing 10% FBS (fetal bovine    serum), and cultured in a 96-well flat bottomed plate at 37° C.    under 5% CO₂ and left stand for 7 days. Culture supernatants from    the wells where colonies had formed were assayed by ELISA    (Enzyme-Linked Immunosorbent Assay) publicly known and commonly used    using Xanthomonas lipopolysaccharide as the antigen. The cells    producing the monoclonal antibody obtained above were cloned by a    limiting dilution method publicly known and commonly used. As a    result, 21 murine monoclonal IgG antibodies against Xanthomonas    lipopolysaccharide were obtained.)-   Subsequently, the PVDF membrane was washed five times with Tris    buffered saline (TBS: 20 mM Tris HCl, pH 7.5, 150 mM NaCl)    containing 0.05% Tween 20. After washing, 5 ml of alkaline    phosphatase-conjugated anti-murine IgM goat immunoglobulin (A9688,    Sigma) diluted 1000 times with PBS containing 1% BSA was added as a    secondary antibody, and the membrane was reacted at room temperature    for 60 minutes. Subsequently, the membrane was washed five times    with TES containing 0.05% Tween 20. After washing, 5 ml of alkali    phosphatase buffer (50 mM Tris HCl, pH 9.5, 1 mM MgCl₂) containing a    chromogenic substrate, 0.0165% 5-bromo-4-chloro-3 indolyl phosphate    (025-08651, Wako Pure Chemical Industries Ltd., Japan) and 0.033%    nitroblue tetrazolium (N-6876, Sigma) was added, and thereafter the    PDVF membrane was transferred into distilled water to stop the    reaction. As a result, LPSx was detected by all of the 21    antibodies, but the limulus-positive glycolipid from xanthan gum did    not react with the 20 antibodies, and reacted with only one    antibody. In this way, it was found that the limulus-positive    glycolipid extracted from xanthan gum has antigenicity which is    different from that of lipopolysaccharide (LPSx) from Xanthomonas.    From this result, it has been shown that the structure of the    limulus-positive glycolipid extracted from xanthan gum is different    from the structure of lipopolysaccharide (LPSx) from Xanthomonas.

5) Example of Functional Food of Limulus-Positive Glycolipid Extractedfrom Xanthan Gum

Production of Candy Containing Limulus-Positive Glycolipid Extractedfrom Xanthan Gum

The mixture as raw materials obtained by adding the limulus-positiveglycolipid extracted from xanthan gum produced in 1) to granulatedsugar, starch syrup and water at a ratio of 5:5:5:1 was heated andboiled down at 120 to 140° C., then cooled on a steel plate for cooling,stretched into a bar-shape and molded into a small round shape of around1 g to produce a candy containing the limulus-positive glycolipidextracted from xanthan gum.

An appropriate amount of this candy was added to 20 ml of water anddissolved by heating. A mass of the limulus-positive glycolipidextracted from xanthan gum in this solution was measured, and the amountwas 6 μg/g. This candy was ingested by 6 males and females who had coldswith a sore throat. Immediately after, a questionnaire regarding thesore throat was conducted. All six persons felt that their respectivethroats had abated (one-sample sign test: p<0.03).

All publications, patents and patent applications cited herein aredirectly incorporated herein by reference.

The invention claimed is:
 1. A concentrated limulus-positive glycolipidobtained by extracting the limulus-positive glycolipid from xanthan gumby adding a polymyxin B immobilized resin to a buffer solutioncontaining the xanthan gum, the concentrated limulus-positive glycolipidhaving a molecular weight of about 2000 to about 4000 Da measured bySDS-PAGE.
 2. A limulus-positive glycolipid composition containing thelimulus-positive glycolipid according to claim
 1. 3. Thelimulus-positive glycolipid composition according to claim 2 whereinsaid limulus-positive glycolipid composition is a pharmaceutical, apharmaceutical for animals, a quasi drug, a cosmetic, a food, afunctional food, a feedstuff or a bath agent.
 4. A concentratedlimulus-positive glycolipid extracted from Xanthan gum.
 5. Thelimulus-positive glycolipid extracted from Xanthan gum according toclaim 4 having a molecular weight of about 2000 Da to about 4000 Dameasured by SDS-PAGE.
 6. The limulus-positive glycolipid extracted fromXanthan gum according to claim 4 having an immunopotentiation effect. 7.The limulus-positive glycolipid extracted from Xanthan gum according toclaim 6 capable of producing NO from murine macrophage lineage cellline.
 8. The limulus-positive glycolipid extracted from Xanthan gumaccording to claim 4 having a structure capable of binding to polymyxinB.
 9. The limulus-positive glycolipid extracted from Xanthan gumaccording to claim 4 having antigenicity different from that oflipopolysaccharide from Xanthomonas microbial cells.
 10. Thelimulus-positive glycolipid extracted from Xanthan gum according toclaim 4 wherein the Xanthan gum is derived from i Xanthomonas.